Novel OrfF and OrfF&#39; polypeptides, nucleic acid molecules encoding the polypeptides and applications thereof

ABSTRACT

The invention relates to novel pathogenic OrfF and OrfF′ polypeptides derived from  Xanthomonas campestris  pv. campstris, the nucleic acid molecules encoding the polypeptides and the uses of the same for detecting or preventing a black-rot disease of a crucifer plant, making organic fertilizer or composting and being a biofilter for degradation of organic compounds.

FIELD OF THE INVENTION

[0001] The present invention relates to novel OrfF and OrfF′polypeptides, nucleic acid molecules encoding the polypeptides, and theapplications thereof.

BACKGROUND OF THE INVENTION

[0002] Composting is the biological conversion of organic wastes, suchas vegetable refuses, woodchips, leave litters or food wastes, intovaluable products, such as fertilizers, substrates for growing mushroom,or biogas (methane) for use as energy sources. In comparison withchemical fertilizers, organic fertilizers are less expensive and havemany agricultural advantages. For instances, soil modified with compostsor organic fertilizers showed improvement of total porosity, increase ofwater stable aggregates (Nnabude, P. C., and Mbagwu, J. S., 2001,Bioresour. Technol., 76:265-272) and accumulation of metals in soil(Guerrero, et al., 2001, Bioresour. Technol., 76: 221-227; and Zinati,et al., 2001, J. Environ. Sci. Health B. 36: 229-243). Crop yield wasenhanced and the growth period thereof was shortened (Ferrer, et al.,2001, Bioresour. Technol. 76: 39-44; Nnabude and Mbagwu, supra; andGuerrero, et al., supra). Termine, et al. found that leeks and turnipsgrown under organic fertilizations had less nitrate contents than thosegrown under inorganic fertilizations (Termine, et al., 1987, PlantsFoods Hum. Nutr. 37:321-332).

[0003] Moreover, compost-modified soil could suppress occurrence ofdiseases on growing plants (Wuest, P. J., and Forer, L. B., 1975,Mycopathologia 55: 9-12; Kannangara, et al., 2000, Can. J. Microbiol.46: 1021-1028). Therefore, the amounts of pesticides and fungicides usedcan be reduced or eliminated. In addition, since soil organisms can bekilled by these pesticides and fungicides, it is considered thatcomposts or organic fertilizers are environmentally safe and capable ofretaining soil fertility. In fact, the soil modification with composthas been demonstrated as an effective method in remediation ofcontaminated soil (Vouillamoz, J., and Mike, M. W., 2001, Water Sci.Technol. 43: 291-295; Semple, et al., 2001, Environ. Pollut. 112:269-283).

[0004] During composting, the active component mediating thebiodegradation and conversion is the resident microbial community. As acomposing process proceeds, the microbial community changes. Forinstance, some microbes were enriched and some were eliminated duringthe process (Peters, et al., 2000, Appl. Environ. Microbiol. 66:930-936).

[0005] For many households or companies, plant leaves constitute themain portion of the starting materials for making organic fertilizers orcomposting. Crucifer plants are the most important vegetables worldwide,including Brassica chinensis, broccoli, cabbage, cauliflower, Brusselssprouts, Chinese cabbage, kale, radish, turnip and mustard. Leaves ofthe crucifers are either edible or discarded. Xanthomonas campestris pv.campstris is a bacterial pathogen of crucifer plants. It infects theleaves of the plants through natural openings (stomata and hydathodes)or wounds due to insect bites, resulting a black-rot disease of theplants (Williams, P. H., 1980, Plant Dis. 64: 736-742).

[0006] In addition, a compost-based biofilter for degradation of organiccompounds have also been successfully developed (Lee, et al., 1999, J.Air Waste Manag. Assoc. 49: 1068-1074; Juteau, et al., 1999, Appl.Microbiol. Biotechnol. 52: 863-868). The biofilter is beneficial for theindustry and the environment, such as bioremediation of hazardous wastesites, biofiltration of industrial water or air and forming a biobarrierto protect soil and ground water from contamination.

[0007] Our earlier studies showed that a spontaneous avirulent mutant ofX. campestris pv. campstris strain 11(Xc11), which was called Xc11A, waslikely resulted from transposition of a specific copy of insertionsequence IS1478a (Chen, et al., 1999, J. Bacteriol., 181: 1220-1228)located in the genome of Xc11 to a position of 352 bp downstream (Hsiau,S. L., 1996, thesis, National Chung Hsing University). It is desired toisolate the black rot gene from Xc11 or the related strains and obtain agene product useful in degradation of organic plant materials in a fast,efficient, simplified, controllable and environmentally safe manner.

SUMMARY OF THE INVENTION

[0008] In one aspect, the invention provides a novel OrfF polypeptidecomprising an amino acid sequence of SEQ ID NO: 1 and the functionalequivalents thereof, and a novel OrfF′ polypeptide comprising an aminoacid sequence of SEQ ID NO: 3 and the functional equivalents thereof. Inone embodiment, the OrfF polypeptide is derived from X. campestris pv.campstris strain 11 (Xc11) and the OrfF′ polypeptide is derived from X.campestris pv. campstris strain 17 (Xc17).

[0009] In another aspect, the invention provides an orfF nucleic acidmolecule encoding the OrfF polypeptide of the invention, and thedegenerate sequences thereof, and an orfF′ nucleic acid moleculeencoding the OrfF′ polypeptide of the invention, and the degeneratesequences thereof. In one embodiment, the orfF nucleic acid moleculecomprises a nucleotide sequence of SEQ ID NO: 2 and the orfF′ nucleicacid molecule comprises a nucleotide sequence of SEQ ID NO: 4.

[0010] In another aspect, the invention provides a recombinant vectorcomprising the nucleic acid molecule of the invention and a regulatorysequence operatively linked thereof. In addition, the invention providesa recombinant cell or organism transformed with the nucleic acidmolecule or the recombinant vector of the invention. Furthermore, theinvention provides a method for preparing the polypeptide of theinvention, comprising the steps of culturing the recombinant cell ororganism of the invention under the conditions suitable for expressingthe polypeptide, and recovering the polypeptide from the culture.

[0011] In still another aspect, the invention provides a method fordetecting a black-rot disease of a crucifer plant, comprising the stepsof providing a sample of a crucifer plant and treating the sample withthe nucleic acid molecule of the invention as a probe under conditionssuch that the nucleic acid molecule can hybridize with a native orfF ororfF′ nucleic acid molecule in the sample. The invention furtherprovides a method for preventing the development of a black-rot diseaseof a crucifer plant, comprising the steps of providing an antisensenucleic acid fragment of the orfF or orfF′ nucleic acid molecule of theinvention and applying an effective amount of the antisense nucleic acidfragment to the crucifer plants.

[0012] In another aspect, the invention provides a method for preparinga recombinant crucifer plant resistant to a block-rot disease,comprising transforming a crucifer plant with an antisense nucleic acidfragment of the nucleic acid molecule of the invention. The inventionfurther provides a recombinant crucifer plant resistant to a block-rotdisease, which is prepared by the above method.

[0013] In another aspect, the invention provides an antibody directed tothe polypeptide of the invention. The invention further provides amethod for detecting a black-rot disease of a crucifer plant, comprisingthe steps of providing a sample of a crucifer plant and treating thesample with the antibody of the invention as a probe whereby theantibody reacts with a native OrfF or OrfF′ polypeptide in the sample.The invention further provides a method for preventing the developmentof a black-rot disease of a crucifer plant, comprising the steps ofapplying an effective amount of the antibody of the invention to thecrucifer plant.

[0014] In still another aspect, the invention provides a process formaking organic fertilizers or composting, comprising the steps ofproviding an organic starting material, adding the OrfF or OrfF′polypeptide of the invention into the organic staring material to form amixture, and incubating the mixture under conditions suitable forforming organic fertilizers or compost.

[0015] In still another aspect, the invention provides a biofilter fordegradation or removal of organic compounds, comprising a filter supportand the OrfF or OrfF′ polypeptide of the invention or a recombinant cellor organism expressing the polypeptide distributed on the filtersupport.

[0016] Other aspects of the present invention will become apparent fromthe following descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1: The nucleic acid sequence of SEQ ID NO: 2 encoding theOrfF polypeptide of Xc11 and the amino acid sequence of SEQ ID NO: 1deduced by the nucleic acid sequence of SEQ ID NO: 2. Amino acidresidues are shown by single letter codes.

[0018]FIG. 2: Southern hybridization of BamHI-EcoRI restricted genomicDNAs of Xc11 and its orfF::Km^(r) knockout mutant using a 0.35-kb orfFDNA fragment as probe. Lane 1, Xc11; lane 2, the orf::Km^(r) knockoutmutant; lane M, HindIII-restricted λ DNA fragments with sizes indicatedon the left.

[0019]FIG. 3: Autoradiograph of ³⁵S-labelled cellular proteins ofplasmid-containing BL21(DE3) pLysS cells separated by SDS-PAGE. Lanes 1and 2, pET21b::orfF-containing BL21(DE3) pLysS cells; lanes 3 and 4,pET21b-containing BL21(DE3) pLysS cells; Lanes 1 and 3, without IPTGinduction; lanes 2 and 4, with IPTG induction. Sizes of maker proteinsare indicated on the left.

[0020]FIG. 4: SDS-PAGE and Coomasie blue staining of cellular proteinsof plasmid-containing DH1(DE3) cells. Lanes 1 and 2,pET21b::orfF-(His)6-containing DH1(DE3) cells; lanes 3 and 4,pET21b-containing DH1(DE3) cells; lane M, protein size markers withsizes indicated on the left. Lanes 1 and 3, without IPTG induction;lanes 2 and 4, with IPTG induction.

[0021]FIG. 5: SDS-PAGE and Coomasie blue staining of proteins inflow-through and the first three eluents during purification of theOrfF-(His)6 protein by affinity chromatography. Lane 1, total cellularproteins from uninduced cells; lane 2, total cellular proteins fromIPTG-induced cells; lane 3 and 4, flow-throughs; lane 5, 6 and 7, thefirst three eluents; lane M, protein size markers with sizes indicatedon the left.

[0022]FIG. 6: Western hybridization of the total cellular proteins inIPTG-induced culture of the pET21b::orfF-(His)6-containg DH1(DE3) cells(lane 1) and the proteins in the eluent during purification of theOrfF-(His)6 protein by affinity chromatography (lane 2), using anti-Hisantibody as probe.

[0023]FIG. 7: SDS-PAGE and Coomasie blue staining of the HPLC-purifiedprotein from culture of the pET21b::orfF-(His)6-containg DH1(DE3) cells.Lane 1, total cellular proteins from uninduced cells; lane 2, totalcellular proteins from IPTG-induced cells; lane 3, proteins in eluent ofthe HPLC protein peak. Lane M, protein size markers with sizes indicatedon the left.

[0024]FIG. 8: Western hybridization of total proteins and culturalmedium proteins from cultures of Xc17, Xc11 and the orfF::Km^(r)knockout mutant of Xc11 using antibody against OrfF-(His)6 protein as aprobe. Lanes 1-3, total proteins from the culture of Xc17 (lane 1), Xc11(lane 2) and the orfF::Km^(r) knockout mutant of Xc11 (lane 3); lanes4-6, cultural medium proteins from the culture of Xc17 (lane 4), Xc11(lane 5) and the orfK::Km^(r) knockout mutant of Xc11 (lane 6).

[0025]FIG. 9: Gus and DAPI stains of onion epidermal cells bombardedwith pBI221 and its derivatives. A and B, pBI221; C and D, BI221containing orfF; E and F, pBI221 containing orfF with deletion of thenucleotides encoding the three consecutive lysine residues in theOrfF-GUS protein; G and H, pBI221 containing orfF with mutation of thenucleotides encoding the three consecutive lysine residues in theOrfF-GUS protein. A, C, E and G, Gus stains; B, D, F and H, DAPI stains.

[0026]FIG. 10: The black-rot symptom in a leaf vein of Brassicachinensis after inoculation with buffer containing the OrfF-(His)6protein. The buffer without the OrfF-(His)6 protein served as control.Sites of inoculation are indicated by arrows.

[0027]FIG. 11: The nucleic acid sequence of SEQ ID NO: 4 encoding theOrfF′ protein of Xc17 and the amino acid sequence of SEQ ID NO: 3deduced by the nucleic acid sequence of SEQ ID NO: 4. Amino acidresidues are shown by single letter codes.

[0028]FIG. 12: Symptoms after injecting the cultural medium of Xc17 orthe orfF::Km^(r) knockout mutant of Xc11 into leaf veins of Brassicachinensis. (A) 6 days after injection with the cultural medium of theorfF::Km^(r) knockout mutant of Xc11; (B) 6 days after injection withthe cultural medium of Xc17; (C) 12 days after injection with thecultural medium of the orfF::Km^(r) knockout mutant of Xc11; (D) 12 daysafter injection with the cultural medium of Xc17.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Due to concern about the potential for enrichment and possibletransfer of pathogens into the food chain as well as to better controlthe compost quality, we have been looking for microbial proteins thathave biodegradation capabilities with broad substrate spectrum. In thestudy, we successfully isolated novel genes from the genomic DNAs ofXc11 and Xc17, a closed-related virulent strain to Xc11, based on theabove-mentioned 352-bp DNA fragment, and obtained polypeptides encodedby the genes capable of biodegradation of a plant body.

[0030] Accordingly, the invention provides an OrfF polypeptidecomprising an amino acid sequence of SEQ ID NO: 1 and an OrfF′polypeptide comprising an amino acid sequence of SEQ ID NO: 3. In oneembodiment of the invention, the OrfF polypeptide is prepared from Xc11and the OrfF′ polypeptide is prepared from Xc17. The polypeptide of theinvention is capable of inducing a black-rot disease of a crucifer plantand has a biodegradation activity on plant materials.

[0031] The OrfF or OrfF′ polypeptide of the invention comprises thefunctional equivalent of the same. In other words, the functionalequivalent of the OrfF or OrfF′ polypeptides of the invention are withinthe scope of the invention. As used herein, the “functional equivalent”of a polypeptide may contain one or more amino acid mutations (e.g.,deletion, addition or substitutions) that result in silent changes onthe corresponding amino acid codon and do not substantially affect thefunction of the polypeptide, such as the induction of a black-rotdisease of a crucifer plant or the biodegradation activity on plantmaterials. For instance, the polypeptide of the invention comprising anamino acid D (aspartic acid) located in a certain position of the aminoacid sequence is functionally equivalent to that comprising an aminoacid E (glutamic acid) at the corresponding position since the two aminoacids, D and E, are both classified as acid amino acids and have similarcharacteristics. More detailed technologies used to obtain thepolypeptide of the invention and the functional equivalent thereof aredescribed below.

[0032] In another aspect, the invention provides an orfF nucleic acidmolecule encoding the OrfF polypeptide and the functional equivalentsthereof, and an orfF′ nucleic acid molecule encoding the OrfF′polypeptide and the functional equivalents thereof. In one preferredembodiment of the invention, the orfF nucleic acid molecule comprises anucleotide sequence of SEQ ID NO: 2 and the orfF′ nucleic acid moleculecomprises a nucleotide sequence of SEQ ID NO: 4. The orfF nucleic acidmolecule of the invention can be isolated from the Xc11 genomic DNAs andthe orfF′ nucleic acid molecule of the invention can be isolated fromthe Xc17 genomic DNAs. The nucleic acid sequences of the presentinvention can be engineered using methods generally known in the art inorder to alter the OrfF or OrfF′ encoding sequences for a variety,including but not limited to, alterations which modify the cloning,processing, and/or expression of the gene product.

[0033] The orfF or orfF′ nucleic acid molecule of the inventioncomprises the degenerate sequences of the same. In other words, thedegenerate sequences of the orfF or orfF′ nucleic acid molecule arewithin the scope of the invention. “Degenerate sequences” of a nucleicacid molecule, as described herein, may contain one or more nucleotidemutations (e.g., deletion, addition or substitutions) that do notsubstantially affect the function of the nucleic acid molecule, such asencoding the polypeptide of the invention or hybridizing with a nativeorfF or orfF′ gene in a plant sample. The term “a native orfF or orfF′gene in a plant sample” as used herein refers to a natural orfF or orfF′gene or the fragment thereof derived from a pathogenic bacterium, suchas Xc11 or Xc17, contained in a sample of a plant. The term “a nativeOrfF or OrfF′ polypeptide” as used herein refers to a natural OrfF orOrfF′ polypeptide or the fragment thereof encoded by a native orfF ororfF′ gene or the fragment thereof. More detailed technologies used toobtain the nucleic acid molecules of the invention and the functionalequivalent thereof are described below.

[0034] In addition, the invention provides a recombinant vectorcomprising the nucleic acid molecule as set forth above and a regulatorysequence operatively linked thereto. The term “vector” used hereinrefers to a nucleic acid molecule capable of carrying and transferring anucleic acid fragment of interest into a host cell for the purpose ofexpression or replication of the same. In particular, a vector refers toa plasmid, cosmid, bacteriophage or virus. Typically, the nucleic acidfragment of interest is operatively linked to a regulatory sequence suchthat, when introducing into a host cell, for instance, the nucleic acidfragment can be expressed in the host cell under the control of theregulatory sequence. The regulatory sequence may comprise, for example,a promoter sequence (e.g., cytomegalovirus (CMV) promoter, simian virus40 (SV40) early promoter and T7 promoter), replication origin and othercontrol sequences (e.g., Shine-Dalgano sequences and terminationsequences). Preferably, the nucleic acid fragment of interest may beconnected to another nucleic acid fragment such that a fused polypeptide(e.g., His-tag fused polypeptide) is produced and is beneficial to thesubsequent purification procedures. The method for identifying andselecting the regulatory sequences is well known to persons skilled inthe art and widely described in the literatures. The skilled persons canreadily construct the recombinant vector of the invention according tothe specification and the well-known literatures.

[0035] The recombinant vector or the nucleic acid molecule of theinvention can be introduced into a host cell or an organism to produce arecombinant cell or organism for the expression of the polypeptideencoded by the nucleic acid molecule of the invention. A suitable hostcell or organism can be derived from a plant, animal, bacterium (e.g.,E.coli), fungus (e.g., yeast), insect, protozoa, virus, mycoplasma, etc.According to the conventional technologies in this art, persons skilledin the art can prepare a suitable recombinant vector and choose asuitable host cell or organism to express and isolate the polypeptide ofthe invention. Accordingly, the invention provides a method forpreparing the polypeptide of the invention, comprising the steps ofculturing the recombinant cell or organism as described above under theconditions suitable for expressing the polypeptide of the invention, andrecovering the polypeptide of the invention from the culture. Thepolypeptide prepared by the method can be further purified by aconventional process (e.g., HPLC or a affinity column). Therefore, therecombinant cell or organism and the method for preparing thepolypeptide are within the scope of the invention. Preferred embodimentsof the host cell or organism and more detailed steps for conducting themethod to obtain the polypeptide of the invention are described below.

[0036] The genetic engineering methods mentioned above such as DNAcloning, vector construction, transformation, protein expression, andpurification can be accomplished by those skilled in this art, and whichcan be seen, for example, in Molecular Cloning: A Laboratory Manual, 2ded., Cold Spring Harbor Laboratory Press, Sambrook, J., E. F. Fritschand T. Maniatis eds. (1989).

[0037] According to the invention, the OrfF or OrfF′ polypeptide iscapable of inducing a black-rot disease of a crucifer plant, and thenative orfF and orfF′ genes encoding the OrfF and OrfF′ polypeptides,respectively, derived from X. campestris pv. campstris, are consideredas pathogenic genes for inducing the black-rot disease. Accordingly, thenucleic acid molecule of the invention can be used as a probe to detecta black-rot disease of a crucifer plant by a hybridization-basedtechnology. The invention provides a method for detecting a black-rotdisease of a crucifer plant, comprising the steps of providing a sampleof a crucifer plant and treating the sample with the nucleic acidmolecule of the invention as a probe under conditions such that thenucleic acid molecule can hybridize with a native orfF or orfF′ gene inthe sample. In one preferred embodiment, the sample is derived fromleaves of the crucifer plant to be detected. Preferably, the cruciferplant is Brassica chinensis, broccoli, cabbage, cauliflower, Brusselssprouts, Chinese cabbage, kale, radish, turnip or mustard. Thehybridization technology used in the method of the invention is wellknown in the art, such as Southern or Northern hybridizationtechnologies as described by Sambrook et al., 1989, Molecular Cloning.

[0038] An antisense nucleic acid fragment is a single-stranded nucleicacid molecule (preferably less than 30 bases) having a sequencecomplementary to certain regions of a target gene and forming a hybridduplex with the target gene by hydrogen-bonded base pairing. Thishybridization can disrupt expression of both the mRNA and the proteinencoded by the target gene. An antisense nucleic acid fragment is wellknown as a tool to inhibit the expression of a target gene (e.g., apathogenic gene) and to enhance the resistance of a plant to pathogens.As mentioned above, Xc11 and Xc17 contain pathogenic genes orfF andorfF′ of the black-rot disease in a crucifer plant. It is useful toprovide an antisense nucleic acid fragment to inhibit the expression ofthe pathogenic orfF and orfF′ genes and prevent the development of ablack-rot disease of a crucifer plant. Accordingly, the inventionprovides a method for preventing the development of a black-rot diseaseof a crucifer plant, comprising the steps of providing an antisensenucleic acid fragment of the orfF or orfF′ nucleic acid molecule of theinvention and applying an effective amount of the antisense nucleic acidfragment to the crucifer plant. In one preferred embodiment, theantisense nucleic acid fragment is applied to leaves of the cruciferplant, preferably, Brassica chinensis, broccoli, cabbage, cauliflower,Brussels sprouts, Chinese cabbage, kale, radish, turnip or mustard. Thesynthesis of an antisense nucleic acid fragment of a target gene is wellknown in the art. Persons skilled in the art can synthesize a suitableantisense nucleic acid fragment of the orfF or orfF′ nucleic acidmolecule of the invention based on the disclosure of the specificationin combination with the conventional technologies, such as thosedescribed in Sambrook et al., supra.

[0039] In addition, the antisense nucleic acid fragment of the orfF ororfF′ nucleic acid molecule of the invention can be introduced into acrucifer plant to provide a resistance to a block-rot disease for thecrucifer plant. Accordingly, the invention provides a method forpreparing a recombinant crucifer plant resistant to a block-rot disease,comprising transforming a crucifer plant with an antisense nucleic acidfragment of the nucleic acid molecule of the invention. A recombinantcrucifer plant resistant to a block-rot disease prepared by the abovemethod is also within the scope of the invention. In one embodiment ofthe invention, the crucifer plant is Brassica chinensis, broccoli,cabbage, cauliflower, Brussels sprouts, Chinese cabbage, kale, radish,turnip or mustard. Preferably, the recombinant crucifer plant isresistant to a block-rot disease caused by Xc11 or Xc17.

[0040] In another aspect, the invention provides an antibody directed tothe OrfF or OrfF′ polypeptide of the invention. The polypeptide of theinvention can be used as an immunogen to prepare an antibody directed toit. The OrfF or OrfF′ polypeptide is purified as described above andintroduced into a suitable animal, such as a rabbit or mouse, and theresultant antibody in the serum are collected, isolated and purified.The resultant antibody is a polyclonal antibody having a specificbinding affinity to the OrfF or OrfF′ polypeptide. Alternatively, theOrfF or OrfF′ polypeptide can be used to prepare a monoclonal antibodyagainst it by using a hybridoma technology well known in the art. In onepreferred embodiment of the invention, a purified OrfF polypeptide isinjected into a mouse in a suitable amount to generate an antibodyagainst the OrfF polypeptide. The antibody can specifically bind to theOrfF polypeptide in an effective titer, such as 1:5000, preferably1:10,000 and most preferably 1:20,000.

[0041] Due to the specificity, the antibody of the invention is usefulin the detection of a black-rot disease of a crucifer plant.Accordingly, the invention provides a method for detecting a black-rotdisease of a crucifer plant, comprising the steps of providing a sampleof a crucifer plant and treating the sample with the antibody of theinvention as a probe under conditions whereby the antibody reacts with anative orfF or orfF′ polypeptide or the fragment thereof in the sample.

[0042] As described above, the OrfF or OrfF′ polypeptide of theinvention can induce a black-rot disease of a crucifer plant and isuseful in biodegradation of an organic starting material (e.g., leavesof the crucifers) for making organic fertilizers or composting.Accordingly, the invention provides a process for making organicfertilizers or composting, comprising the steps of providing an organicstarting material containing a plant body, adding the OrfF or OrfF′polypeptide of the invention into the organic starting material to forma mixture, and incubating the mixture under the conditions suitable forforming organic fertilizers or compost. In a preferred embodiment, theorganic starting material may be vegetable refuses, woodchips, leavelitters or food wastes. The plant body contained therein is preferablyderived from leaves of a crucifer plant, such as Brassica chinensis,broccoli, cabbage, cauliflower, Brussels sprouts, Chinese cabbage, kale,radish, turnip and mustard. The process for making compost, andmaterials, extracts, biochemicals or biogases thus produced as the end-or by-product through the process is within the scope of the invention.The OrfF and OrfF′ polypeptides of the invention or the recombinant cellor organism expressing the OrfF and OrfF′ polypeptides, and materials,extracts, biochemicals or biogases as described above can be used asintegrants of feed, folder, medium, manure, compost, fertilizer ornutritional modifications or supplements for cultivation or feeding,killing, inactivation or restricting the growth of living organisms; asintegrants of a soil conditioner or for biomediation of soil to improvethe condition and fertility of the soil or modify a contaminated soil;and as integrants of fumigants or energy sources.

[0043] It is known that a biofilter in supporting specificmicroorganisms is capable of significantly degrading sulfur compoundsand hydrocarbon vapors. Hydrogen sulfide, methyl mercaptan, and dimethyldisulfide have been successfully degraded using a biofiltrationtechnique at the concentrations observed in wastewater treatment plantand paper-pulp mill fugitive emissions. Studies were also directed tothe volatile organic compounds such as n-butane, benzene, and toluene.Benefits of using a biofiltration technology include economy ofinstallation and operation, simplicity of maintenance, and ability totreat co-pollutants. Due to the biodegradation activity, the OrfF andOrfF′ polypeptides of the invention or the recombinant cells ororganisms expressing the OrfF and OrfF′ polypeptides can be incorporatedinto a biofilter for degradation of organic compounds and isadvantageous for the environmental safety. Accordingly, the inventionprovides a biofilter for degradation or removal of organic compounds,comprising a filter support and the OrfF and OrfF′ polypeptides of theinvention or a recombinant cell or organism expressing the OrfF andOrfF′ polypeptides distributed on the filter support.

EXAMPLES

[0044] The present invention will become apparent with reference to thebelow examples. The examples described below are given by way ofillustration only and not intended to be any limitation to the presentinvention.

Example 1

[0045] Genomic DNA of Xc11 was extracted from a 35 ml overnight cultureand 200 g of genomic DNA was obtained. About 0.2 g of Xc11 genomic DNAwas used as template to PCR-amplify the 352-bp DNA fragment with theprimer pairs 352-L (5′-TAATAACACTCCTTGC-3′) and Xc11A-R(5′-CTCGGATCCCTCCATCTTCTCCTGA-3′). The PCR fragment was gel-purified,radiolabelled with (α-³²P)dCTP and used as a probe to screen about 4000phage plaques from an Xc11 genomic library stock according to the methoddescribed by Sambrook et al., supra (Southern hybridization). Fourpositive bacteriophage clones were found. Of them, one clone was pickedfor further analysis. The phage DNA was prepared and restriction-mappedaccording to the method described by Sambrook et al., supra. A 2.6-kbEcoRI-BamHI DNA fragment of the phage DNA that included the 352-bpregion was cloned into plasmid pUC18.

Example 2

[0046] The 2.6-kb EcoRI-BamHI DNA fragment was cloned into plasmid pUC18(Yanish-Perron, et al., 1985, Gene 33: 103-119) and the nucleotidesequence was determined with universal forward and reverse primers.Within the sequence, only one orf (open reading frame) was found.Database search revealed that the orf did not show sequence homologywith any known genes. It was named as orfF. FIG. 1 shows the nucleotidesequence of orfF and the deduced amino acid sequence of the putativeOrfF polypeptide.

Example 3

[0047] A 1.3-kb BamHI Km^(r) cassette from plasmid pUC4K (AmershamPharmacia Biotech) was cloned into the SspI site of the orfF gene in the2.6-kb EcoRI-BamHI DNA fragment and the resulting 3.9-kb EcoRI-BamHIfragment was cloned into suicide vector pSUP202 (Simon, et al., 1983,Bio/technology 2: 784-791). The recombinant plasmid was introduced toXc11 via triparental mating (Ditta, et al., supra) and Km^(r)transconjugants were selected. Genomic DNAs of the transconjugants wereextracted, restricted with EcoRI and BamHI, and Southern hybridizationwas performed using PCR-amplified 0.35-kb orfF DNA fragment described inExample 1 as a probe. One transconjugant that had successful replacementof the chromosomal 2.6-kb orfF fragment with the 3.9-kb orfF::Km^(r)fragment was picked (FIG. 2). The pathogenicity of this orfF::Km^(r)knockout mutant was examined according to the method described by Danielet al., 1984, J. Gen. Microbiol. 130: 2447-2455, and the results showedthat the knockout mutant did not elicit any rotting symptom with any ofthe 8 test turnip seedlings. It was concluded that the orfF gene wasresponsible for the rotting capability of Xc11.

Example 4

[0048] The orfF gene DNA fragment was PCR-amplified with plasmid pTcαand primer pairs L (5′-TGCTCTAGACGCCAAATTCAGAAAAGC-3′) and R1(5′CCCAAGCTTTTAATTAAATGCTTCCGC-3′), and gel-purified. The orfF DNAfragment was cloned into the XbaI and HindIII sites of plasmid pET21b(Novagen) and transformed into E. coli BL21(DE3) pLysS (Novagen).Expression of the orfF gene in the transformant was examined accordingto the method modified from the methods of Tabor and Richardson, 1985,Proc. Natl. Acad. Sci. USA. 82: 1074-1078 and Ajdic and Ferretti, 1998,J. Bacteriol. 180: 5727-5732. Basically, 1 mM IPTG was added into 5 mlof mid-log phase culture and incubation was continued for 1.6 hour,which was followed by ³⁵S-Methionine labeling for 10 minutes. The cellswere harvested by centrifugation and dissolved in 100 μl of SDSgel-loading buffer, of which 15 μl was used for SDS-PAGE analysis andautoradiography. As shown in FIG. 3, the orfF gene was expressed as a 13kd protein with the culture of the pET21b::orjF-containing cells, butnot with the culture of the pET21b-containing cells. Therefore, the orfFgene could be expressed as a 13 kd OrfF protein in vivo by the T7promoter in pET21b.

Example 5

[0049] For the purpose of generating an OrfF-(His)6 fusion protein andan antibody against the protein, the orfF gene DNA fragment wasPCR-amplified and cloned into the NdeI and HindIII sites of plasmidpET21b (Novagen) so that a Shine-Dalgano sequence was located in frontof the orfF gene and a (His)6-tag sequence was linked to the C-terminalend of the expressed OrfF protein, and the plasmid was transformed intoDH1(DE3) (laboratory stock). For induction of the OrfF-(His)6 protein, 1mM IPTG was added to the mid-log phase culture of the transformant andincubation was continued for 2 hours. Cell pellets were harvested andthe total cellular proteins were analyzed by SDS-PAGE. As shown in FIG.4, the cells harboring pET21b::OrfF-(His)6 showed an over-expression ofa protein with the same size as expected for the OrfF-(His)6 protein (14kd) after IPTG induction. On the other hand, cells harboring pET21b didnot show induction of proteins of similar sizes.

Example 6

[0050] Purification of the OrfF-(His)6 protein from culture ofpET21b::orfF-(His)6-containing cells was performed according to themethods described in the pET System manual (Novagen, 9^(th) edition).Basically, 1 mM IPTG was added to 50 ml of the mid-log phase culture ofthe pET21b::OrfF-(His)6-containing DH1(DE3) cells and incubation wascontinued for 2 hours. Cell pellets were harvested and cell extract wasprepared by sonication. Inclusion body in the cell extract was collectedthrough several centrifugation and washing steps and about 100 mg ofinclusion body was obtained. The OrfF-(His)6 protein in the inclusionbody was purified by the method modified from the method of Shi et al.,1997, Biotechniques 23: 1036-1038. The pellet was first dissolved in 2ml of the binding buffer (20 mM Tris, 0.5 M NaCl, 5 mM imidazol, and 8 Murea; pH 7.8) completely and 2 ml of Ni-NTA agarose (Qiagen) was added.After incubation overnight at 4° C., the mixture was packed in an emptycolumn and washed with 5 volumes (10 ml) of the binding buffer first andlater 5 volumes (10 ml) of the wash buffer (20 mM Tris, 0.5 M NaCl, 20mM imidazol, and 8 M urea; pH 7.8). Flow-through from the wash bufferwas collected. Three volumes (6 ml) of the elution buffer (20 mM Tris,0.5 M NaCl, 0.3 M imidazol, pH 7.8) was then applied and the eluent wascollected into a tube every 1 ml. As a result, 6 tubes of eluent werecollected. For the first 3 tubes containing the eluent from the elutionbuffer and the 2 tubes containing the flow-through from the wash buffer,15 μl each was taken for SDS-PAGE analysis. As shown in FIG. 5, aprotein band of 14 kd was observed with the 5 samples examined.Solutions in the 6 tubes containing the eluent from the elution bufferwere pooled and 5 μl was used for SDS-PAGE analysis and probing withanti-His antibody (Invitrogen) (Western hybridization). Total cellularproteins from IPTG-induced culture of the pET21b::orfF-(His)6-containingcells was analyzed together as control. As shown in FIG. 6, ahybridization signal corresponding to a protein of 14 kd was observedwith the eluent and the total cellular proteins of the IPTG-inducedculture. Thus, the eluent contained only one 14-kd protein with a (His)6tag in the sequence. The purified protein was likely the OrfF-(His)6protein..

Example 7

[0051] To confirm that the protein purified in Example 6 was indeed theOrfF-(His)6 protein, the protein in the eluent in Example 6 was furtherpurified by HPLC (High Performance Liquid Chromatography) and subjectedto N-terminal sequencing. The total of approximate 6 ml of eluent inExample 6 was loaded onto a C18 column (5C-18-Ms, Cosmosil) and elutedwith an acetonitrile gradient (0% to 60% acetonitrile in 1%trifluoroacetate). A protein peak was observed and the protein wascollected as a 2 ml solution. The solution was concentrated into 50 μlby Centricon 10 (Millipore) and 10 μl were analyzed by SDS-PAGE. Totalcellular proteins of both IPTG-induced and uninduced cultures ofpET21b::orfF-(His)6-carrying DH1(DE3) cells were analyzed together forcomparison.. As shown in FIG. 7, a single protein band corresponding tothe 14-kd OrfF-(His)6 protein was observed with the HPLC-purifiedprotein sample and the total cellular proteins of the IPTG-inducedculture. The remaining 40 μl HPLC-purified protein solution wassubjected to N-terminal sequencing. The result indicated that the first5 amino acid residues of the purified protein are the same as thoseexpected from the OrfF protein sequence (FIG. 1). This result and theresult from Example 6 clearly indicated that protein purified by theprocedures in Example 6 was indeed the OrfF-(His)6 protein.

Example 8

[0052] The OrfF-(His)6 protein prepared according to the procedures inExample 4 was quantitated by the protein assay kit (Bio-rad). About 5 μgof the protein was used to immunize a mouse in order for generation ofantibody against the OrfF-(His)6 protein. The antibody was used as1:20000 dilution for probing the cell extracts from both IPTG-inducedand uninduced cultures of pET21::orfF-(His)6-carrying DH1(DE3) cells,and a hybridization signal corresponding to the OrfF-(His)6 protein wasobserved. 250 ml of the cultures of Xc11, Xc17, a virulent strainclosely related to Xc11, and the orfF::Km^(r) knockout mutant of Xc11were grown in secretion medium (Rossier, et al., 1999, Proc. Natl. Acad.Sci. USA. 96: 9368-9373) and were checked for production and secretionof the OrfF protein according to the method described by Rossier, etal., 1999, supra. To prepare the total protein fractions, proteins ofthe 5 ml cultures of the both cells were TCA-precipitated, and dissolvedin 100 μl SDS gel-loading buffer. To prepare the culture medium proteinfractions, the remaining 200 ml cultures of both cells were centifugatedand the cell-free supernatants were filtered through a 0.22 μm filter(GPWP04700, Millipore). The proteins in the filtrates wereTCA-precipitated and dissolved into 500 μl SDS gel-loading buffer.Fifteen μl of total protein fractions and culture medium proteinfractions of the three cells were used for SDS-PAGE analysis, followedby probing with anti-OrfF-(His)6 antibody (Western hybridization). Asshown in FIG. 8, hybridization signals were observed with both thecultural medium proteins and the total proteins from culture of Xc11 andanother virulent strain Xc17, but not with those from culture of theorfF::Km^(r) knockout mutant of Xc11. However, the hybridization signalscorrespond to a protein of 21 kd in size, instead of 13 kd which is thesize of OrfF protein in Xc11 as detected in Example 4.

Example 9

[0053] The orfF gene DNA fragment was PCR-amplified and cloned into theXbaI and SmaI sites of plant expression plasmid pBI221 (Clontech), inwhich expression of the OrfF-GUS fusion protein was under the control ofCaMV 35S promoter. The recombinant plasmid was introduced into onionepidermal cells via particle bombardment according to the methoddescribed by Varagona et al., 1992, Plant Cell 3: 105-113. Gus stainingwas then performed according to the method described by Varagona et al.,1992, supra and the cells were observed under a light microscope. It wasfound that blue stains were localized in the nucleus of the cellsbombarded with the orfF gene-carrying pBI221 plasmid. In contrast, bluestains were observed in the cytoplasm of cells bombarded with pBI221(FIG. 9). The cells were then stained with a nucleic acid stain, DAPI,and observed under fluorescence microscope (Varagona, et al., 1992). Thelight blue fluorescence stains co-localized with the Gus stains in cellsbombarded with the orfF gene-carrying pBI221 plasmid, but not with thosein cells bombarded with pBI221 (FIG. 9). This indicated that theOrfF-GUS protein was capable of entering nuclei of the plant cells,whereas the GUS protein could stay in the cytoplasm of the plant cells.Site-directed mutagenesis was performed with the orfF gene-carryingpBI221 plasmid so that the three lysine residues at 28^(th), 29^(th),and 30^(th) residues in the OrfF sequence were either deleted or changedinto three threonine residues. The resulting two mutant plasmids wereagain bombarded into onion cells followed by Gus and DAPI stains. Theresults showed that the Gus stains were observed in the cytoplasm of theonion cells with the two mutant plasmids (FIG. 9). It was thus concludedthat OrfF protein, when introduced into plant cells, could enter plantnucleus.

Example 10

[0054] The OrfF-(His)6 protein was purified according to the methoddescribed in example 6, except that the last elution step was replacedby the following renaturation and elution steps. After washing with fivevolumes of wash buffer as described in Example 6, the column was washed9 times each with 10 ml of binding buffers containing either 8 M, 7 M, 6M, 5 M, 4 M, 3 M, 2 M and 1 M urea in order and, lastly, 10 ml of bidingbuffer without urea. Three volumes (6 ml) of elution buffer were appliedand about 5 ml of eluent was collected. Protein concentration in theeluent was determined by protein assay kit (Bio-rad), which was 700 ngper μl. A test shown in FIG. 10, black-rot symptom was observed in theinjection site with the OrfF-(His)6 protein-containing elution buffer,but not with the elution buffer only. Therefore, OrfF-(His)6 proteinalone was capable of rotting leaves of Brassica chinensis.

Example 11

[0055] Primer pairs pLXC11F4 (5′-CAACGTGTTCCGTCC-3′) and PTCaL2(5′-GATCAACACCAATTACGC-3′) corresponding to the sequences upstream ofIS1478a and downstream of orfF in Xc11 were used to amplify thecorresponding region in Xc17. A 2.5-kb DNA fragment was obtained, clonedand sequenced. The 2.5-kb sequence was found to be identical to theexpected 4.0 kb sequence in the corresponding DNA region of Xc11, exceptthat the IS1478a copy located upstream of orfF in Xc11 and its adjacent5-bp sequence were deleted in Xc17. The deletion resulted in generationof a new open reading frame by in-frame addition of 246-bp sequence 5′to orfF. This new orf is called orfF′. The OrfF′ protein not only can begenerated and secreted in Xc17 but also in Xc11, which should be due tospontaneous excision of the IS1478a copy and the adjacent 5-bp sequencein Xc11. The OrfF protein could not be generated in the orJF::Km^(r)knockout mutant of Xc11. FIG. 11 shows the nucleotide sequence of orfF′and the deduced amino acid sequence of the putative ORfF′ protein.

Example 12

[0056] One-hundred-and-twenty ml of cultures of Xc17 and theorfF::Km^(r) knockout mutant of Xc11 were grown in secretion medium(Rossier, et al., 1999, Proc. Natl. Acad. Sci. USA. 96: 9368-9373) andconcentrated by Centricon (Millipore). Protein concentrations weredetermined by protein assay kit (Bio-rad) wherein 19.32 μg per ml forXc17 and 20.59 μg per ml for the knockout mutant. Fifty μl of each wasapplied to leaf veins of Brassica chinensis through a 1-ml syringe. Asshown in FIG. 12, black-rot symptom was observed in the injection sitewith the cultural medium of Xc17, but not with that of the knockoutmutant. Therefore, the OrfF′ protein in the cultural medium of Xc17 wascapable of rotting leaves of Brassica chinensis.

What is claimed is:
 1. An OrfF polypeptide comprising an amino acidsequence of SEQ ID NO: 1 and the functional equivalents thereof.
 2. TheOrfF polypeptide of claim 1, which is derived from Xanthomonascampestris pv. campestris strain 11 (Xc11).
 3. A nucleic acid moleculeencoding the OrfF polypeptide of claim 1, and the degenerate sequencesthereof.
 4. The nucleic acid molecule of claim 3, which comprises anucleotide sequence of SEQ ID NO:
 2. 5. A recombinant vector comprisingthe nucleic acid molecule of claim 3 and a regulatory sequenceoperatively linked thereto.
 6. A recombinant cell or organismtransformed with the nucleic acid molecule of claim 3 or the recombinantvector of claim
 5. 7. The recombinant cell or organism of claim 6, whichis derived from a plant, animal, bacterium, fungi, insect, protozoa,virus or mycoplasma.
 8. A method of preparing the OrfF polypeptide ofclaim 1, comprising the steps of culturing the recombinant cell ororganism of claim 6 under the conditions suitable for expressing theOrfF polypeptide, and recovering the OrfF polypeptide from the culture.9. A method for detecting a black-rot disease of a crucifer plant,comprising the steps of providing a sample of the crucifer plant andtreating the sample with the nucleic acid molecule of claim 3 as a probewhereby the nucleic acid molecule can hybridize with a native orfFnucleic acid molecule in the sample.
 10. The method of claim 9, whereinthe sample is derived from leaves of the crucifer plant.
 11. The methodof claim 9, wherein the crucifer plant is Brassica chinensis.
 12. Amethod for preventing the development of a black-rot disease of acrucifer plant, comprising the steps of providing an antisense nucleicacid fragment of the nucleic acid molecule of claim 3 and applying aneffective amount of the antisense nucleic acid fragment to the cruciferplant.
 13. The method of claim 12, wherein the antisense nucleic acidfragment is applied to leaves of the crucifer plant.
 14. The method ofclaim 12, wherein the crucifer plant is Brassica chinensis.
 15. A methodfor preparing a recombinant crucifer plant resistant to a block-rotdisease, comprising transforming a crucifer plant with an antisensenucleic acid fragment of the nucleic acid molecule of claim
 3. 16. Themethod of claim 15, wherein the block-rot disease is caused by Xc11. 17.The method of claim 15, wherein the crucifer plant is Brassicachinensis.
 18. A recombinant crucifer plant resistant to a block-rotdisease, which is prepared by the method of claim
 15. 19. Therecombinant crucifer plant of claim 18, wherein the block-rot disease iscaused by Xc11.
 20. The recombinant crucifer plant of claim 18, which isBrassica chinensis.
 21. An antibody directed to the OrfF polypeptide ofclaim
 1. 22. A method for detecting a black-rot disease of a cruciferplant, comprising the steps of providing a sample of the crucifer plantand treating the sample with the antibody of claim 21 as a probe wherebythe antibody reacts with a native OrfF polypeptide in the sample. 23.The method of claim 22, wherein the sample is derived from leaves of thecrucifer plant.
 24. The method of claim 22, wherein the crucifer plantis Brassica chinensis.
 25. A method for preventing the development of ablack-rot disease of a crucifer plant, comprising the steps of applyingan effective amount of the antibody of claim 21 to the crucifer plant.26. The method of claim 25, wherein the antibody is applied to leaves ofthe crucifer plant.
 27. The method of claim 25, wherein the cruciferplant is Brassica chinensis.
 28. A process for making organicfertilizers or composting, comprising the steps of providing an organicstarting material containing a plant body, adding the OrfF polypeptideof claim 1 into the organic starting material to form a mixture, andincubating the mixture under the conditions suitable for forming organicfertilizers or compost.
 29. A process of claim 28, wherein the organicstarting material is selected from the group consisting of vegetablerefuses, woodchips, leave litters and food wastes.
 30. A process ofclaim 28, wherein the plant body is derived from leaves of a cruciferplant.
 31. The process of claim 30, wherein the crucifer plant isBrassica chinensis.
 32. A biofilter for degradation or removal oforganic compounds, comprising a filter support and the OrfF polypeptideof claim 1 or a recombinant cell or organism expressing the OrfFpolypeptide distributed on the filter support.
 33. The biofilter ofclaim 32, wherein the organic compounds are selected from the groupconsisting of hydrogen sulfide, methyl mercaptan, dimethyl disulfide,n-butane, benzene, and toluene.
 34. An OrfF′ polypeptide comprising anamino acid sequence of SEQ ID NO: 3 and the functional equivalentsthereof.
 35. The OrfF′ polypeptide of claim 34, which is derived fromXanthomonas campestris pv. campestris strain 17 (Xc17).
 36. A nucleicacid molecule encoding the OrfF′ polypeptide of claim 34, and thedegenerate sequences thereof.
 37. The nucleic acid molecule of claim 36,which comprises a nucleotide sequence of SEQ ID NO:
 4. 38. A recombinantvector comprising the nucleic acid molecule of claim 3 and a regulatorysequence operatively linked thereto.
 39. A recombinant cell or organismtransformed with the nucleic acid molecule of claim 36 or therecombinant vector of claim
 38. 40. The recombinant cell or organism ofclaim 39, which is derived from a plant, animal, bacterium, fungi,insect, protozoa, virus or mycoplasma.
 41. A method of preparing theOrfF′ polypeptide of claim 34, comprising the steps of culturing therecombinant cell or organism of claim 39 under the conditions suitablefor expressing the OrfF′ polypeptide, and recovering the OrfF′polypeptide from the culture.
 42. A method for detecting a black-rotdisease of a crucifer plant, comprising the steps of providing a sampleof the crucifer plant and treating the sample with the nucleic acidmolecule of claim 36 as a probe whereby the nucleic acid molecule canhybridize with a native orfF′ nucleic acid molecule in the sample. 43.The method of claim 42, wherein the sample is derived from leaves of thecrucifer plant.
 44. The method of claim 42, wherein the crucifer plantis Brassica chinensis.
 45. A method for preventing the development of ablack-rot disease of a crucifer plant, comprising the steps of providingan antisense nucleic acid fragment of the nucleic acid molecule of claim36 and applying an effective amount of the antisense nucleic acidfragment to the crucifer plant.
 46. The method of claim 45, wherein theantisense nucleic acid fragment is applied to leaves of the cruciferplant.
 47. The method of claim 45, wherein the crucifer plant isBrassica chinensis.
 48. A method for preparing a recombinant cruciferplant resistant to a block-rot disease, comprising transforming acrucifer plant with an antisense nucleic acid fragment of the nucleicacid molecule of claim
 36. 49. The method of claim 48, wherein theblock-rot disease is caused by Xc17.
 50. The method of claim 48, whereinthe crucifer plant is Brassica chinensis.
 51. A recombinant cruciferplant resistant to a block-rot disease, which is prepared by the methodof claim
 48. 52. The recombinant crucifer plant of claim 51, wherein theblock-rot disease is caused by Xc17.
 53. The recombinant crucifer plantof claim 51, which is Brassica chinensis.
 54. An antibody directed tothe OrfF′ polypeptide of claim
 34. 55. A method for detecting ablack-rot disease of a crucifer plant, comprising the steps of providinga sample of the crucifer plant and treating the sample with the antibodyof claim 54 as a probe whereby the antibody reacts with a native OrfF′polypeptide in the sample.
 56. The method of claim 55, wherein thesample is derived from leaves of the crucifer plant.
 57. The method ofclaim 55, wherein the crucifer plant is Brassica chinensis.
 58. A methodfor preventing the development of a black-rot disease of a cruciferplant, comprising the steps of applying an effective amount of theantibody of claim 54 to the crucifer plant.
 59. The method of claim 58,wherein the antibody is applied to leaves of the crucifer plant.
 60. Themethod of claim 58, wherein the crucifer plant is Brassica chinensis.61. A process for making organic fertilizers or composting, comprisingthe steps of providing an organic starting material containing a plantbody, adding the OrfF′ polypeptide of claim 34 into the organic startingmaterial to form a mixture, and incubating the mixture under theconditions suitable for forming organic fertilizers or compost.
 62. Aprocess of claim 61, wherein the organic starting material is selectedfrom the group consisting of vegetable refuses, woodchips, leave littersand food wastes.
 63. A process of claim 61, wherein the plant body isderived from leaves of a crucifer plant.
 64. The process of claim 63,wherein the crucifer plant is Brassica chinensis.
 65. A biofilter fordegradation or removal of organic compounds, comprising a filter supportand the OrfF′ polypeptide of claim 34 or a recombinant cell or organismexpressing the OrfF′ polypeptide distributed on the filter support. 66.The biofilter of claim 65, wherein the organic compounds are selectedfrom the group consisting of hydrogen sulfide, methyl mercaptan,dimethyl disulfide, n-butane, benzene, and toluene.